Unveiling an unexpected superoxide-mediated photooxidation mechanism of squalene monohydroperoxides to squalene hydroperoxy cyclic peroxides through ESR and LC–MS/MS analyses

Lipid cyclic peroxides are a rarely reported and documented class of compounds in the human organism. Recently, we reported the formation of squalene (SQ) hydroperoxy cyclic peroxides derived from SQ monohydroperoxide isomers (SQ-OOHs) for the first time. Notably, we successfully detected and quantified cis-2-OOH-3-(1,2-dioxane)-SQ in the human skin. Nevertheless, the underlying mechanism governing the formation of these compounds remained elusive. Therefore, in the current study, we set to determine the reaction’s mechanism. To this end, a comprehensive analysis of the precise conditions involved in the onset and propagation of this conversion was carried out by oxidizing total SQ-OOHs under different conditions, including singlet oxygen (1O2), thermal, and photoinduced oxidations monitored by quantifying the generated 2-OOH-3-(1,2-dioxane)-SQ using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Radical intermediates were thoroughly investigated using Electron Spin Resonance (ESR) with the aid of spin traps and radical references. Moreover, calculations of SQ-OOHs’ electrostatic charges were performed on Spartan 18 software. We found that the reaction is ideally induced and favored under photooxidation in the presence of 3O2 in hexane, and that superoxide radical (O2•−) is the first key intermediate in this mechanism, whereas peroxyl radicals were the major species observed throughout the oxidation. Chemical calculations provided an explanation for the targeting of tertiary SQ-OOHs by this reaction and gave further evidence on the proposed heterolytic cleavage initiating the reaction. The novel oxidation mechanism suggested herein offers new insights into understanding lipid secondary oxidation and is a promising finding for further studying lipid cyclic peroxides in general.


Fig. S1
. 1 O2 generation over 60 min from RB in H2O (A), D2O (B), and isopropanol (IPA) (C), measured by its phosphorescence at 1280 nm (FP8700 apparatus).During the reaction with SQ, RB is dissolved primarily in H2O.D2O was used since the lifetime of 1 O2 is known to be longer in deuterated solvents, permitting the confirmation of the generation of 1 O2 in H2O.Interestingly, the intensity of 1 O2 signal was higher when RB was dissolved in IPA, this information is particularly useful when the reaction's main solvent is not miscible with water, RB can be dissolved in IPA while guaranteeing the generation of an equal, not to say a greater amount of 1 O2.
2-LC-UV analysis of SQ-OOHs in the presence and absence of EP over 72 h.4-LC-UV analysis of EP in the absence and presence of SQ-OOHs over 18 h.6-1 H NMR analyses of EP's decomposition in the absence and presence of SQ-OOHs.
Compatibility between the characteristic peaks of fully decomposed EP can be observed in EP incubated at 25 °C for 18 h, and in EP + SQ-OOHs under the same conditions.Details are presented in the assignments table and spectra below.15-Schematic interpretation of the inductive effect's role in the targeting of tertiary SQ-OOHs compared to secondary SQ-OOHs.The scheme shows the impact of the inductive effect, which was described as an influential factor together with hyperconjugation and the steric effect in the stability of the resulting radicals.

Fig. S2 .
Fig. S2.Changes occurring on SQ-OOHs LC-UV peaks in normal phase in the presence of EP over 72 h.The slight decrease in the intensity of the peaks is believed to be due to the formation of volatile decomposition products arising from SQ-OOHs radical decomposition which is inevitable at room temperature.

Fig. S4 .
Fig. S4.LC-UV chromatograms of EP at 0 h, EP or EP+SQ-OOHs after a prolonged period of 18 h of incubation at 25°C.Peaks from 1 h to 18 h showed the same pattern.

Fig.
Fig. S9. 1 H NMR spectrum of EP at 0 h prior to the start of the incubation.

Fig. S12 .
Fig. S12.Expected decomposition mechanisms of EP and proposed radical-induced decomposition of EP promoted by the presence of trace amounts of alkoxyl and hydroxy radicals resulting from SQ-OOHs' O-O homolytic decomposition upon incubation at 25°C.

Fig. S13 .
Fig. S13.Q1 MS scan of (A1) SQ ozonolysis and (B1) SQ-OOHs ozonolysis for a period of 60 S. LC-MS chromatograms of the main products observed from (A2) SQ ozonolysis where only 3 species could be identified as spcified in the figure, and (B2) SQ-OOHs ozonolysis for the same period of time.Overall, it can be seen that in both cases, breakdown products were the main produts resulting from the exposure of SQ and SQ-OOHs to ozone.

Fig. S14 .
Fig. S14.Stick diagram interpretation of the hyperfine splitting of DMPO-OR generated from the UV irradiation of tBuOOH (a N =14.29 G and aß H ≈14.21 G).

Fig. S16 .
Fig. S16.Stick diagram interpretation of the hyperfine splitting of DMPO-OO − generated from the quenching of potassium from KO2.

Fig. S20 .
Fig. S20.Schematic representation of the targeting of tertiary SQ-OOHs compared to secondary SQ-OOHs by the described mechanism.The scheme shows the impact of the inductive effect, which was described as an influential factor together with hyperconjugation and the steric effect in the stability of the resulting radicals.

Fig. S22 .
Fig. S22.DMPO radical adducts resulting from SQ-OOHs (A) thermal oxidation, and (B) photooxidation.Under thermal oxidation, a significant number of breakdown species could be observed with the formation of both DMPO-O-SQ and DMPO-OO-SQ, while under photooxidation, there were less breakdown products with one main adduct detected belonging to DMPO-OO-SQ.These observations confirm our proposed hypothesis that under photooxidation, SQ-OOHs' O-H bond is mainly targetted, while under thermal conditions, both O-O and O-H can be cleaved, with the former giving rise to mainly breakdown products.